Assay using recombinant histidyl-tRNA synthetase

ABSTRACT

Recombinant Histidyl tRNA synthetase produced by non-mammalian host cells is used in a sensitive assay to determine the presence of autoimmune diseases in mammals. Methods for isolating, cloning and expressing rHRS are described. In addition, a kit for determining the presence of an autoimmune disease is provided.

BACKGROUND OF THE INVENTION

The aminoacyl-tRNA synthetases constitute a group of about twentyproteins which play a crucial role in translating the genetic code bycatalyzing the reaction joining amino acids to their cognate tRNAs.Despite widespread common functionality, their structures are verydiverse. Primary structures of almost all 20 bacterial and yeastsynthetases have been ascertained. This research has led to therecognition of two major synthetase classes based upon bothcrystallographic and primary structure analysis. These findings havegreatly advanced our understanding of the evolutionary pathways that ledto the functional preservation of synthetases despite their remarkablestructural diversity.

In contrast to the data from lower eukaryotes, far less structuralinformation is available for synthetases from higher eukaryotes. Theonly nucleotide sequences of mammalian synthetases so far available arethose of the glutaminyl-synthetase (Fett, R. et al. (1991) J. Biol.Chem. 266: 1448-1455), aspartyl-synthetase (Jacobo-Molina et al. (1989)J. Biol. Chem. 264: 16608-1612), threonyl-synthetase (Cruzen et al.(1991) J. Biol. Chem. 266: 9919-9923), valyl-synthetase (Hsieh et al.(1991)Biochem. J. 278: 809-816), tryptophanyl-synthetase (Garret et al.(1991) Biochemistry 30: 7809-7817) and histidyl-synthetase (Tsui et al.(1987) Nucleic. Acids. Res. 15: 3349-3367; Tsui et al. (1987) Gene 61:349-361).

The histidyl-tRNA synthetase (HRS) has been particularly interestingsince it is a frequent target of autoantibodies relating to humanautoimmune diseases including myositis, polymyositis and dermatomyositis(Nishikai et al. (1980) Arthritis Rheum. 23: 881-888). Four othersynthetases have also been found as targets ofautoantibodies--threonyl-synthetase (Mathews et al. (1984) J. Exp. Med.160: 42-434), alanyl-synthetase (Bunn et al. (1987) Mol. Biol. Med. 4:21-36), glycyl-synthetase, and isoleucyl-synthetase (Targoff, I. N.(1990) J. Immunol. 144: 1737-1743). However, these synthetases were onlyrarely found to be an autoimmune target.

Patients having these autoantibodies form a distinct group clinically,and perhaps genetically (Love et al. (1991) Medicine (Baltimore) 70:360-374). Recent studies on the mechanisms of anti-HRS induction andregulation suggest that the native human enzyme is the antigen whichselects and sustains the immune response, preceding clinical illness(Miller et al. (1990) J. Clin. Invest. 85: 468-475; Miller et al. (1990)Proc. Natl. Acad. Sci. U.S.A. 87: 9933-9937).

Assays for detecting HRS in serum have been developed (Biswas et al.(1987), Journal of Immunological Methods 98: 243-248) and have provenvery useful for detecting autoimmune diseases related to HRS in humanserum. To produce these assays purified HRS protein was isolated fromHeLa cells using high performance liquid chromatography (HPLC). Theisolated HRS protein was then bound to an ELISA plate and incubated inthe presence of human serum (Biswas et al. (1987), Journal ofImmunological Methods 98: 243-248). Detectable binding of antibodies inthe serum to the bound HRS protein indicated an autoimmune diseasestate.

HRS proteins have also been isolated from calves' liver (Targoff et al.,J. Immunol., 138:2874-2882 (1987). However HRS proteins isolated fromcalves' liver or HeLa cells were very unstable, even when stored at -80°C. For this reason, ELISA experiments using such isolated HRS proteinshad to be performed rapidly following isolation. In addition, isolatingHRS using HPLC is a time-consuming and difficult process.

The HRS gene from a SV40 transformed fibroblast cell line was cloned andexpressed in COS 1 cells (Tsui et al. Nucl. Acid. Res. (1987) 8:3349-3367). Three regions of extensive homology between the Human HRSand a hamster HRS clone were discovered. In addition, Ramsden et al.(Journal of Immunology (1989) 143: 2267-2272) has epitope mapped theexpressed product of a cloned HRS gene. However, in these experimentsthe HRS cDNA was transiently transfected into COS 1 cells which alreadyexpress an endogenous HRS. For this reason, background binding fromexpression of the endogenous enzyme made determining the amount ofbinding from the transfected clone difficult.

Although others have disclosed methods of isolating HRS from HELA cellextracts, these proteins were only weakly immunogenic in assays forautoimmune diseases. It would be very advantageous to provide a HRSwhich was much more sensitive to antibodies than the previouslydisclosed protein.

SUMMARY OF THE INVENTION

One preferred embodiment of the present invention is recombinantHistidyl tRNA synthetase produced by transfection into non-mammalianhost cells, wherein the recombinant Histidyl tRNA synthetase issubstantially free from endogenous Histidyl tRNA synthetase produced inthe host cells.

Another aspect of the present invention is a method for determining thepresence of an autoimmune disease in a mammal. The diseases areadvantageously myositis, polymyositis, or dermatomyositis. This methodincludes isolating a sample of body fluid from a mammal wherein the bodyfluid contains antibodies. Following isolation of body fluid, preferablyblood serum, the fluid can be advantageously stored for greater than 24hours. The body fluid is then contacted with recombinant Histidyl tRNAsynthetase produced in a non-mammalian host cell, preferably an insectcell. This contact is more preferably an ELISA, with the Histidyl tRNAmost preferably being stored at a temperature within the range -80° C.to 25° C. the recombinant Histidyl tRNA synthetase being substantiallyfree of endogenous Histidyl tRNA synthetase from the host cell. Aftercontacting the recombinant Histidyl tRNA synthetase with the body fluidthe presence or absence of binding of the antibodies to the recombinantHistidyl tRNA synthetase is detected, wherein detectable binding of theantibodies to the recombinant Histidyl tRNA synthetase indicates thepresence of an autoimmune disease in the mammal.

Still another embodiment of the present invention is a method fordetermining the presence of an autoimmune disease in a mammal. Theautoimmune disease is preferably polymyositis, myositis anddermatomyositis. In this method a sample of body fluid from a mammal isisolated, with the body fluid having antibodies. The next step of themethod is obtaining a peptide of Histidyl tRNA synthetase with thepeptide being produced in non-mammalian host cells wherein the HistidyltRNA synthetase is substantially free of endogenous Histidyl tRNAsynthetase from the host cell. Preferably the peptide is the first 60amino acids of Histidyl tRNA synthetase. Alternatively, the peptide isthe first 47 amino acids of Histidyl tRNA synthetase. The peptide mostpreferably comprises an amino acid sequence that contains less than thefull-length amino acid sequence of Histidyl tRNA synthetase with thepeptide containing at least one antigenic determinant present infull-length Histidyl tRNA synthetase. The next method step is contactingthe isolated body fluid, preferably blood serum, with the peptide anddetecting the presence or absence of binding of the antibodies to thepeptide of Histidyl tRNA synthetase. Most advantageously, the contactingstep is an ELISA and detectable binding of the antibodies in the bodyfluid to the peptide fragment indicates the presence of an autoimmunedisease in the mammal.

Yet another embodiment of the present invention is a kit for determiningthe presence of antibodies from an autoimmune disease in the blood serumof a mammal. This kit comprises recombinant Histidyl tRNA synthetaseproduced by expression in a non-mammalian host cell, a vessel forperforming an assay to detect binding of the antibodies to therecombinant Histidyl tRNA synthetase, and reagents for detecting thebinding of the antibodies to the recombinant Histidyl tRNA synthetase,wherein positive binding indicates the presence of an autoimmune diseasein the mammal. In this kit the vessel is preferably a microtiter plate,and the mammal is a human. Advantageously, the reagents for detectingthe binding of the antibodies to the recombinant Histidyl tRNAsynthetase comprise labeled anti-human antibodies. In addition, thedetection reagents preferably comprise reagents for performing an ELISA.

DESCRIPTION OF THE FIGURES

FIGS. 1A-1C. Hybridization of amplified HRS DNA with oligonucleotidesspecific for Hep G2 HRS or for the published sequence. FIG. 1A is aschematic diagram of PCR amplified fragments and positions of primersites for PCR. 5' and 3' fragments of the HRS cDNA encompassing theentire coding region were amplified with Taq DNA polymerase from Hep G2HRS cDNA (positive control), IMR 90 and HFM-1 cell lines. Fragmentscorresponding to exon 6 through exon 8 and exons 8 and 10 separatelywere amplified from genomic DNA. FIG. 1B shows a direct sequenceanalysis of PCR-amplified Hep G2 HRS cDNA corresponding to mid-exon 8and mid-exon 10. Boxes represent regions of non-identity between Hep G2and the published sequence. Oligonucleotides specific for Hep G2 (983and 987) or for the published sequence (2491 and 986) were used to probePCR amplification products. Oligonucleotides 2491 and 986 were also usedfor low stringency PCR amplification from genomic DNA to generatepositive controls for the published sequence (lanes 2 and 4). Theamplified DNA hybridized to Hep G2 specific probes. In contrast,oligonucleotides specific for the published sequence hybridized only theDNA amplified in order to generate a positive control.

FIGS. 2A-2B. Alignment of the predicted amino acid sequences of E. coli,yeast, and human HRS. Motifs 1, 2 and 3 (Eriani et al. (1990) Nature347: 203-26) and signature regions 1 and 2 (see Discussion) areindicated above the E. coli sequence. The 32 amino acid shared motif isunderlined. E. coli, yeast, and human are identical; * there is aconserved substitution. Arrows beneath the predicted hamster sequenceindicate amino acids likely to be incorrect based upon conservationamong the others. The alignment was modified by hand from an alignmentproduced by the Clustal program in PCGene.

FIG. 3A-3B. Signature regions. Computer generated alignments weremodified by hand to maximize alignment within an amino acid family.

DETAILED DESCRIPTION

We have discovered that the recombinant histidyl tRNA synthetase (HRS)protein produced in non-mammalian host cells provides unexpectedlysuperior results when used as an antigen in assays for determiningautoimmune diseases in human serum. We have advantageously produced suchrecombinant HRS protein in non-mammalian hosts, such as insect celllines, by inserting the human histidyl tRNA synthetase (HRS) gens into abaculovirus expression system. We have also found that antigenic peptidefragments of this protein have far greater affinity for autoimmuneantibodies in human serum than would have been expected from studies onHRS purified from HeLa cells.

Although, as discussed in the background, others have expressedrecombinant HRS in COS 1 cells, these cells contain an endogenous HRSprotein which interferes with studies relating to the transfected HRSgene. We have found that the HRS produced by insect cells has noantigenic cross-reactivity with the human HRS and therefore provides avery advantageous host for producing large quantities of isolated HRS.As discussed herein the term isolated, when referring to a protein orpeptide, includes those molecules that have been purified to greaterthan their naturally occurring concentration.

To express the HRS antigen for immunologic experiments, and to study thecontrol of its synthesis, we isolated a cDNA encoding human HRS from thehuman hepatoma cell line, HepG2. This sequence is available as AccessionNo. Z11518 in the European Molecular Biology Labs (EMBL) database. ThecDNA sequence, determined by direct sequencing of PCR-amplifiedfragments, was markedly different from the published sequence of thehuman enzyme (Tsui et al. (1987) Nucleic. Acids. Res. 15: 3349-3367).Differences within the coding region included multiple nucleotidesubstitutions, insertions and deletions resulting in frameshifts whichled to a substantial difference in the protein's predicted primarystructure.

Although it was possible that an allelic variation of the HRS gene hadbeen isolated, numerous experiments with specific PCR amplificationdetermined that the human genome only carried one copy of the HRS gene.In addition, analysis of nucleotide sequences from fetal myoblast andfibroblast cell lines confirmed the accuracy of our sequence. Weconcluded that sequencing errors led to publication of the incorrect HRSnucleotide sequence by Tsui et al., Nucl. Acid Res. (1987) 8: 3349-3367.

As a first step in sequencing and expressing a recombinant clone, weisolated a cDNA sequence corresponding to the HRS protein in HepG2cells. The newly determined sequence differed in 48 places, includinginsertions and deletions, from the previously published sequence by Tsuiet al. By sequence specific probes and direct sequencing, we establishedthat only the newly determined sequence was present in genomic DNA.Further, we have sequenced 500 bases upstream of the translation startsite to search for regulatory regions.

The predicted amino acid sequence derived from the cDNA clearlyexhibited all three of the expected motifs recognized in Class 2aminoacyl-tRNA synthetases. Alignment of E. coli, yeast, and mammalianpredicted amino acid sequences for three of the four members of theclass 2a subgroup (his, pro, ser, and thr) exhibited strong preservationof amino acid specific signature regions proximal to motifs 2 and 3.These potentially represented the binding regions for the proximalacceptor step and the amino acid during translation.

The amino acid sequence of the first two exons of human HRS predicted astructure having a 32 amino acid helical motif. This type of motif wasfirst described in human QRS, a class 1 synthetase, and is also found ina yeast RNA polymerase, a rabbit termination factor, and both bovine andhuman WRS. As these proteins are all RNA binding factors it suggeststhat the first two exons of human HRS may contain an RNA binding motif.

DNA Sequence Analysis

As discussed previously, the cDNA sequence we determined by directsequencing of PCR-amplified fragments (without cloning) differedmarkedly from the published HRS sequence obtained from anSV40-transformed human fibroblast line (Tsui et al. (1987) Nucleic.Acids. Res. 15: 3349-3367). The HepG2 HRS coding region has 39nucleotide substitutions, 6 insertions, and 3 deletions resulting inseveral frameshifts compared to the published sequence.

Several possibilities besides sequencing errors could explain thediscrepancy, including the existence of two genes or of alternativelyspliced forms of a single transcript from two tissue sources. To chooseamong these possibilities, we synthesized HRS cDNA from the IMR 90 andHFM-1 fetalmyoblast cell lines. Total RNA was isolated followed by PCRamplification of two overlapping DNA fragments encompassing the entireHRS coding region. To rule out the possibility of PCR carryover, weamplified a genomic region spanning exon 6 through exon 8 which includes2 small introns, according to the intron-exon boundaries published forthe hamster HRS gene (Tsui et al. (1987) Gene 61: 349-361). Finally,exon 8 and exon 10 were separately amplified from human genomic DNA inan attempt to locate the published sequence somewhere in the genome(FIG. 1A). All the PCR primers (except as noted below) were chosen tomatch regions of exact agreement between the sequences but to flankregions of discrepancy.

For each amplification, a single product of the expected size wasobtained. The products were probed with oligonucleotides specific forHepG2 HRS or for the published sequence. (FIGS. 1B and 1C). HepG2 5'frag and 3' frag served as positive controls for slot blot hybridizationanalysis. Two primers (2491 and 986--Table 1) corresponding to thepublished sequence mid-exon 8 and mid-exon 10 were used for PCRamplification at low stringency (45° C. annealing) to generate apositive control for the published sequence.

Only HepG2 HRS-specific oligonucleotides hybridized under stringentconditions with the corresponding DNA sequences. In contrast, onlycontrol PCR products, produced at low stringency with primers containingpublished sequences, would hybridize with the probes specific for thepublished sequence. Hybridization of duplicate gel blots witholigonucleotide probes (Table 1), corresponding to the Hep G2 (probe 18)or the published sequence (probe 19) for the end of exon 7 alsoexhibited signal only for Hep G2 (data not shown). These results clearlyindicated that only the Hep G2 HRS sequence is present in the humangenome. At the conclusion of these experiments, the entire codingsequence of HRS cDNA from cell line HFM-1 was determined by directsequencing and was found to be identical to the Hep G2 sequence.

                                      TABLE 1                                     __________________________________________________________________________    Oligonucleotide Primers for PCR Amplification                                 PRIMER                                                                              SEQ ID                                                                  NUMBER                                                                              NUMBER                      COMMENTS                                    __________________________________________________________________________                UPSTREAM SEQUENCE                                                 3     1     GGCTGGGGCAACCACCGCAG  5' UT                                       984   2     TCTCCTTTGACCTGAGCCTTG beg. exon 10                                12    3     AGAATGAGATGGTGGGAGAGAAGGG                                                                           beg. exon 8                                 10    4     GCCTGAAGATCATGTGCGAGATCC                                                                            mid exon 6                                  983   5     TTGCACCtGAgGTGGCTGAcCGC                                                                             mid exon 8, probe Hep G2                    2491  6     TTGCACCaGAaGTGGCTGAtCGC                                                                             mid exon 8, probe publ.                     2     7     GCAGAGCGTGCGGCGCTGGA  beg. exon 1                                 1102  8     TGCGTGGGGAGACATCCGGGG intron 1                                    2736  9     GAGGCTCTCTAGGCGTGCG   90 bp 5' to the ATG                                     DOWNSTREAM SEQUENCE                                               4     10    CAGACTGGACTAAGCCTCCTGGGCC                                                                           3' UT                                       16    11    TGGGTTCTTCTTGTACAGCAGG                                                                              exon 11                                     985   12    AGTCTCTGTTCCACGATGGAGAA                                                                             end exon 10                                 17    13    CCACCATGGTTGCTGGACATAGTC                                                                            end exon 8                                  987   14    ACACTGCCCACAcCCAgGGGCTC                                                                             mid exon 10, probe HEP G2                   986   15    ACACTGCCCACA-CCA-GGGCTC                                                                             mid exon 10, probe publ.                    18    16    TgTCcAGcTTGTCtACTGAG  end exon 7, probe HEP G2                    19    17    TaTCcAGcTTGTCtACTGAG  end exon 7, probe publ.                     421   18    CTTGGGGGTTTTGAGCACAAATT                                                                             end exon 2                                  2597  19    CCACTTGAGCCGCCTGCTGTCT                                                                              9 bp 5' to the ATG                          2755  20    GGCTACTAAGGGAACTTGGG  158' bp 5' to the ATG                       __________________________________________________________________________

Analysis of the sequence differences between the Hep G2 gene and thepublished sequence revealed that the changes we discovered in the HRSsequence were not random. At the nucleotide level Hep G2 HRS sharedsubstantially more similarity with yeast HRS (Natsoulis et al. (1986)Cell 46: 235-243) than the Tsui et al. sequence (47.5% versus 40.9%).The sequence between the first in-frame methionine (ATG) codon and thestop codon in the cDNA encodes a 1527 bp open reading frame of 509 aminoacids. The protein encoded by this open reading frame would have anapproximate molecular weight of 57,410 daltons.

The predicted amino acid sequence of the human HRS shares considerablymore homology with yeast HRS than the previously published sequence,particularly in the three structural motifs recently identified in Class2 synthetases (FIG. 2). Among 37 amino acids in motif 1, eighteen areidentical and six are conservative substitutions between yeast andhuman. In this same region, four amino acids are identical and eightconservatively substituted among E. coli (Freedman et al. (1985) J.Biol. Chem. 260: 10063-10068), yeast, and human. Among thirty-six aminoacids in motif 2, thirty are identical and four are conservativesubstitutions between yeast and human. In addition, nine amino acids areidentical and eleven conservatively substituted among E. coli, yeast,and human. In motif 3, among twenty-eight amino acids, fifteen areidentical and six conservatively substituted between yeast and human.Also in motif 3, seven amino acids are identical and sevenconservatively substituted among E. coli, yeast, and human. The threespecies share 78 identical and 89 conservatively substituted amino acidsfor an overall homology of 31.7 percent. In the motif regions, thehydrophobicity plots of the three proteins, determined by the techniqueof Kyte and Doolittle with the SOAP program in PC/GENE using a window of11 amino acids, are nearly superimposable (data not shown).

5' Untranslated Region

To obtain a previously unknown sequence 5' to the translation startsite, we performed inverted PCR. We first amplified and directlysequenced intron 1 of the HRS gene to find a convenient restrictionsite. Primer pairs 2 and 421 (Table 1) corresponding to the beginning ofexon 1 and the end of exon 2 were used for this amplification. A twostep protocol was employed to obtain a previously unknown 530 bpsequence upstream from the translation start site, taking advantage ofthe sequence information on intron 1. Ava I digestion of genomic DNAfollowed by amplification with primers 2597 and 1102 (Table 1) yieldedan approximately 200 bp fragment outside the boundaries of knownsequence. Based on the sequence of this fragment, we designed primers2755 and 2736 to amplify, by inverse PCR, an additional 330 bp ofupstream sequence using the Taq I restriction enzyme. To confirm theidentity of the upstream sequence obtained by inverse PCR, we carriedout genomic amplification of the region, including part of exon 1 inaddition to the 5' untranslated region. This PCR product was directlysequenced with Taq DNA polymerase using the DNA Sequencing System(Promega) and was found to be identical with the sequence obtained byinverse PCR.

Mapping of Transcription Initiation

To determine the 5' end of the HRS mRNA isolated from the Hep G2 andHeLa cell lines, we used primer extension analysis with two different ³²p labeled oligonucleotide primers complementary to the sequence fromnucleotide +4 to +27 and from -137 to -158. In each case, one majorextended product was observed in two cell lines, suggesting that thetranscription initiates 378 to 382 nucleotides behind the ATG startcodon. Two different P-labelled oligonucleotide primers hybridizing 240and 400 nucleotides from the transcription start site were used forprimer extension analysis. The primers were annealed to total RNA fromHep G2 and HeLa cells and extended by reverse transcriptase. The lengthof reverse transcripts was determined on a sequencing gel. A sequenceladder was obtained by using the primer extension ologonucleotide -137to -158 as a sequencing primer and PCR-amplified genomic fragment as atemplate. The position of the start site was determined from asequencing reaction initiating from the same primer using amplifiedgenomic DNA as a template. Several smaller extended products, observedonly with the primer hybridizing downstream from the 5' end of thecoding sequence, most likely represent false start sites due tosecondary structure in the mRNA. The sequence upstream from the startsite did not contain the TATA or CCAAT motifs. Instead, potentialbinding sites for the transcription factor SP1 were found in the5'-flanking region.

Genomic PCR Amplifications

PCR grade human DNA was prepared from whole blood as previouslydescribed (Higuchi, R. (1989) in PCR Technology. Principles andApplications for DNA Amplification, Erlich, H.A. Ed. pp. 31-38,Stockton, N.Y.). Primers 10 and 17 (Table 1 and FIG. 1a) were used toamplify a genomic region covering exons 6-8 that included the two smallintrons. Amplification was performed with 2.5 U of Taq polymerase (1 minat 94° C., 1 min at 65° C., and 2 min at 72° C. for 30 cycles).

Two sets of primers were used to separately amplify exons 8 and 10 fromgenomic DNA. Amplification was carried out with primer pairs 12 and 17(Table 1) for exon 8 (1 min at 94° C., 1 min at 60° C., and 1 min at 72°C.; 30 cycles) and primer pairs 984 and 985 for exon 10 (1 min at 94°C., 1 min at 65° C. and 2 min at 72° C.; 30 cycles) Products of genomicPCR amplifications were used for hybridization as discussed below.

Slot-Blot Hybridization

PCR products (200 ng DNA) were heat-denatured (5 min, 100° C.), dilutedto 200 μl with 6X SSC (0.9 M NaCl, 90 mM sodium citrate, pH 7.0), and100 μl aliquots were applied to a Nytran filter (Schleicher and Shuell,Inc.) pre-soaked in 6X SSC using a slot-blot apparatus. The filter wasbaked for 1 hour at 80° C. Single strips were hybridized to ³²P-labelled oligonucleotides specific to either the Hep G2 HRS or to thepublished HRS sequence by Tsui et al. Hybridization continued overnightat 42° C. in 5X SSPE (0.75 M NaCl, 50 mM NaH₂ PO₄, 5 mM EDTA), 5XDenhardt's solution, and 0.5% SDS, followed by washing several times atroom temperature in 2X SSPE, 0.5% SDS and a 30 minute wash at 55° C. in2X SSPE, 0.5% SDS.

Inverse PCR

To amplify a previously unknown regions upstream from the translationstart site of the HRS gene, we employed the inverse PCR procedure(Triglia et al. (1988) Nucleic. Acids. Res. 16: 8186). Two sets ofenzymes were used for chromosome walking. Genomic DNA (3 μg) was firstcleaved with Ava I and ligated with T4 DNA ligase in a dilute DNAsolution (<3 μg/ml). The resulting circular molecules were cut at aninternal site with Bgl I and amplified with primers 2597 and 1102(Table 1) (1 min at 94° C., 1 min at 60° C. and 2 min at 72° C., 30cycles). Genomic DNA was then cut with Taq I, ligated, re-cut with HglA1and amplified with primers 2755 and 2736 (Table 1) under the conditionsdescribed above. PCR products were directly sequenced by well knownmethods.

Primer Extension

Synthetic oligonucleotides complementary to the HRS mRNA wereend-labelled with T4 polynucleotide kinase, hybridized to total RNA fromHep G2 or HeLa cell lines and extended with reverse transcriptase. A 20μl mixture containing 50 μg RNA, 1.5×10⁵ cpm of ³² P-labelled primer,80% formamide, 0.4M NaCl, 40 mM PIPES (pH 6.4), and 1 mM EDTA wasdenatured at 80° C. for 5 min and hybridized at 33° C. overnight. Thehybridized RNA/DNA was precipitated with ethanol, resuspended in 20 μlof reverse transcription buffer (50 mMTris HCl (pH 8.3), 50 mM KCl, 6 mMMgCl₂. 1 mM DTT, 1 mM each dNTP, 1 U/μl RNAsin, 25 μg/μm l actinomycinD, and 20 U AMV reverse transcriptase (Life Sciences, Inc.) andincubated at 41° C. for 1 hour. The reaction was stopped with 1 μ l 0.5MEDTA and the RNA was hydrolyzed with 2 μl 2N NaOH at 37° C. for 30minutes. The extension products were extracted, ethanol-precipitated,separated on a 6% polyacrylamide/8M urea gel, and detected byautoradiography.

The sequence of human HRS we have determined allows HRS to fitcomfortably with the Class 2 synthetases, unlike the previouslypublished Tsui et al. sequence.

Multiple errors in the previously published human HRS cDNA sequence,including both nucleotide substitutions and frameshifts, led to an 8.9%difference at the amino acid level. Alignment of the sequences of E.coli, yeast, and human (Hep G2) alongside the predicted sequence for thepublished hamster sequence (Tsui (1987) Gene. 61: 349-361) is shown inFIGS. 2A and 2B. The hamster sequence diverges from human in areasconserved between yeast and human and even in areas conserved among theE. coli, yeast and human, suggesting that it, too, contains errors.Areas likely to contain sequencing errors are indicated by arrowsbeneath the hamster sequence in FIG. 2.

The determination of the human HRS sequence, along with the recentlycompleted human TRS sequence (Cruzen et al. (1991) J. Biol. Chem. 266:9919-9923), now allows sequences of Class 2a synthetases for the sameamino acid from a prokaryote (E. coli), a lower eukaryote (yeast), and ahigher eukaryote (human) to be compared. From the published analyses ofClass 2 synthetases--first, by Eriani, et al. (Nature (1990) 347:203-26), and more recently by Cusak, et al. (Nucleic. Acids. Res. (1991)19: 3489-3498)--it has been possible to recognize those structuralelements that have been preserved during the presumed horizontalevolution of a primitive parent synthetase to the ten enzymes whichcomprise the family. The crystal structures of two of these synthetases,including one with the cognate tRNA bound, has allowed certainrecognition of the ATP binding site and of the region which binds the 3'CCA end common to every tRNA. Motif 2 and Motif 3 are concerned withbinding the stem and the ATP, respectively. Because the overall chemicalreaction catalyzed by these enzymes involves the esterification of thecarboxyl group of an amino acid first to ATP, and then to a hydroxyl onthe ribose of the terminal adenosine, these are just the features whichmust be conserved during both the horizontal evolution--from a parentprotein to progeny for ten different amino acids--and the verticalevolution--from species to species.

There are, however, additional requirements for vertical evolution. Theamino acid cavities for the tRNA recognition motifs need to beevolutionarily preserved, along with the anti-codon and the most distalbase pair(s) of the acceptor stem (Hiraki et al. (1987) Kaku. Igaku. 24:1483-1489) have pointed out the region between Motifs 1 and 2 thatencompass loop L1, beta sheet B2 and helix H9 contain some amino acidspecific sequences as does the region encompassing two strands of theactive site beta sheet, βA3, and βA4. The three HRS sequences, the threeTRS sequences and the E. coli and yeast SRS sequences--three of the fourmembers of Class 2a--allow a clearer view of these two regions, whichlie outside the motifs, in which strong vertical preservation suggeststhat they are regions conserved for these purposes.

In FIG. 3a, the region just proximal to motif 2, designated signatureregion 1, is aligned for these sequences. The strong verticalpreservations within all three families is evident. For HRS, among 42amino acids, 10 are identical, 12 are conservatively replaced, and threeruns of three amino acids are conserved. For TRS, among 48 amino acids,20 are identical, 5 are conservatively replaced and there are runs ofnine, seven, and four conserved amino acids. For SRS, among 55 aminoacids, 15 are identical, 11 are conservatively replaced, and there isone run of five and four runs of three conserved amino acids, althoughintroduction of two gaps is necessary. What is striking, however, isthat whereas in motif 2 it is easy to align all of the eight sequences,in signature region 1, the three groups are wholly different.

The alignment of a second area, designated signature region 2, is shownin FIG. 3B. This region is comprised of the β strands A3 and A4 in E.coli SRS. Here the amino acid conservation is even stronger. Allowingfor a single variable gap in the middle of each HRS, among 32 aminoacids, 18 are identical and seven are conservatively replaced. Inaddition, there are conserved runs of eight, six, five and three aminoacids. In TRS, with allowance for a gap in E. coli and human, among 57amino acids, 22 are identical and 11 are conservatively replaced. Also,there are conserved runs of four, five, six and six amino acids. In SRS,among 59 amino acids, 24 are identical and 13 are conservativelyreplaced, including a conserved run of ten, four runs of four, and tworuns of three conserved amino acids. Again, the three groups differstrikingly from one another, but in each group preservation almostalways exceeds preservation within the motifs.

Based on the crystal structures of the closely related class 2asynthetase, E. coli SRS and of the less closely related class 2b yeastDRS (Ruff et al. (1991) Science 252: 1682-1689) these signature regionsare likely to interact with the proximal acceptor stem and the aminoacid, respectively. The low overall homology between some E. coli andyeast pairs and the abnormal charging of a yeast tRNA within E. colihave suggested the possibility that a synthetase might have changed itscommitment to a particular amino acid during evolution. The existence ofthe vertically preserved signature regions shown in FIG. 4 is thestrongest evidence so far of the continuous evolutionary relatedness offamily members.

The amino terminal 60 amino acids in human HRS, which precede Motif 1and are in a region known to vary greatly among synthetases even withinthe same family, has been discovered by Fett and Knippers to have asignificant homology to a 57 amino acid motif which is repeated threetimes in the middle of the anomalously large human QRS (Fett, R. andKnippers, R. (1991) J. Biol. Chem. 266: 1448-1455). The location of thisshared motif, which is wholly contained within the first two exons ofHRS, raises the possibility that the two most proximal exons and thefirst intron of HRS shuffled to QRS. What adds particular interest tothis possibility is the fact that QRS is a Class 1 enzyme whose gene islocated on human chromosome 1 and HRS is a Class 2 enzyme whose gene islocated on human chromosome 5. A recent search of Genbank and EMBL bythe TFasta program of the Wisconsin GCG Sequence Analysis SoftwarePackage, carried out by the Advanced Scientific Computing Facility ofthe Frederick Cancer Research Center, however, shows that the center ofthis 60-amino acid stretch (aa residues 14 to 45--FIG. 2) hassubstantial homology to a variety of other known genes.

Besides human QRS and hamster (but not yeast) HRS, yeast RNA polymeraseC-40 gene (Accession number M15499), rabbit eukaryotic release factor(Accession number m33460), and both bovine (Accession numbers X52113,M74074, J05334, X53918) and human (Accession number M61715) tryptophanyltRNA synthetases are closely related. These are all proteins which bindor interact with RNA, suggesting the possibility, reinforced by thepredicted helical structure, that this is a nucleic acid binding motif.

Following the determination and analysis of the cloned DNA fragment, weexpressed the Hep G2 gene in a baculovirus expression system, andperformed assays for human autoimmune diseases.

We and others had developed detection methods for HRS using purifiedHela cell HRS in an ELISA, but the procedures were expensive anddifficult to perform. Since we were interested in studying the originsof autoimmunity, in particular focussing on autoimmune muscle diseases(myositis), we decided to research antibody binding to recombinant HRS.

Expression of HRS in Baculovirus Expression System

Total RNA was isolated from Hep G2 cells using the RNAzol method(Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156-159).cDNA was synthesized from 2.5 μg of total RNA in a 20 μl reactioncontaining 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂ (1X PCRbuffer, Perkin-Elmer-Cetus), 0.25 mM dNTP, an oligo(dT) primer, and 10 Uof AMV reverse transcriptase (Boehringer, Mannheim). After incubating at42° C. for one hour, reactions were heated at 65° C. for 10 min, and 2μl was used for PCR.

PCR reactions were carried out in a final vol of 100 μl and contained 1XPCR buffer, 200 μM dNTP, 250 ng each of oligonucleotide primers flankingthe coding region (Table 1---primers 3 and 4) and 2.5 U of Taqpolymerase (Perkin-Elmer-Cetus) (1 min at 94° C., 1 min at 55° C., 3 minat 72° C. for 40 cycles). The oligonucleotides were constructed from thepublished HRS-human sequence obtained from an SV 40 transformed humanfibroblast line (Tsui, F. W. and Siminovitch, L. (1987) Nucleic. Acids.Res. 15: 3349-3367). The resulting 1.6 kB DNA fragment was directlysequenced using the protocol we described previously (Raben et al.(1991) Diabetes 40: 118-122). Overlapping sequences were obtained forboth strands of the cDNA.

RNA from human fibroblast line, IMR 90, and human fetal myoblast line,HFM-1, (the kind gift of Dr. Lori Love) was reverse-transcribed using anoligo(dT) or primer 4 (Table 1) and subsequently amplified for 40 cycleswith primer pairs 3 and 16 or 4 and 984 (Table 1 and FIG. 1A) to obtaintwo overlapping DNA fragments ("5' frag" and "3' frag") encompassing theentire coding region of HRS. Each PCR cycle consisted of 1 min at 94°C., 1 min at 68° C., and 2 min at 72° C. DNA fragments from IMR 90 andHFM-1 cell lines were used for hybridization analysis. 5' frag and 3'frag from the Hep G2 cell line served as controls for these experiments.

From PCR-amplified DNA, a 1.6 kb Spe 1 fragment containing the full HRScoding region was obtained and ligated into plasmid pBlueBac®(Invitrogen), linearized with Nhel and dephosphorylated with calfintestinal alkaline phosphatase (CIAP). This plasmid vector has a strongpolyhedron promoter to direct the synthesis of foreign gene products.Clones of pBlueBac-HRS having the HRS cDNA in the correct orientationwere identified by standard methods, followed by plasmid purification.The resulting construct encoded a non-fusion protein with initiation atthe HRS internal ATG, after homologous recombination with AcPNV, theinsect baculovirus.¹²

To obtain recombinant virus, Sf9 cell monolayers were co-transfectedwith the recombinant vector (pBlueBac-HRS) and wild type baculovirus,purchased as linearized viral DNA from Invitrogen, by the calciumphosphate precipitation technique. 5×10⁶ Cells were seeded in 25 cm²flasks in 4 ml of TNM-FH medium with 10% fetal calf serum (GibcoBRL/Life Technologies) and allowed to attach for 1 hour. 2 μg of linearAcNPV was mixed with 4 μg of pBlueBac-HRS in 0.75 ml of transfectionbuffer (25 mM hepes, pH 7.1, 140 mM NaCl, 125 mM CaCl₂). The DNAsolution was added dropwise to the cells containing 0.75 ml freshmedium. After 4 hours, the medium was replaced with 5 ml of freshcomplete medium and the cells were incubated at 27° C. Thevirus-containing inoculum was collected after 7 days and recombinantbaculovirus was purified by plaque assay according to the method of Webband Summers, with diluted transfection supernatants and 1% agaroseoverlay containing X-gal at 150 μg/ml. The recombinant plaques wereeasily distinguishable by blue coloration and then selected by theirocclusion-negative phenotype. Putative recombinant viral isolates werepurified through two rounds of plaque purification to obtain purerecombinant virus stocks. To confirm that isolates contained HRS DNA,genomic viral DNA was prepared according to Webb et al. (Webb, AC, etal., BioTechniques (1991) 11: 512-519) and amplified by PCR with insectspecific oligonucleotide primers (Invitrogen) spanning the entire codingregion of HRS. DNA was prepared from pelleted viral particles and thenamplified with appropriate oligonucleotides by PCR. All tested isolateswere positive for the presence of the insert sequence corresponding tothe HRS cDNA.

The level of recombinant protein expression in baculovirus was assayedby Coomassie blue staining and autoradiography of metabolically labelledcell lysates after SDS-PAGE. For metabolic radiolabelling of recombinantprotein, cells were seeded in a 24 well plate at a density of 0.5×10⁶cells/well, infected at a 5-10 multiplicity of infection (MOI) andincubated at 27° C. for various times. The cells were then incubated for1 hour in methionine-free medium, and for the next 4 hours in mediumcontaining 5 μg, ³⁵ S-methionine.

For production of recombinant protein, cells were infected at 1-10 MOIin 20 ml and incubated for 1 hour. Infected cells were seeded in 100 mlspinner flasks at a density of 2×10⁶ cells/ml. Cultures were grown atroom temperature and 55 rpm and then harvested between 44 and 48 hourspost infection. Cells were washed twice with ice-cold PBS (7,3 mM NaH₂PO₄, 55 mM KCl, 74mM NaCl, 6.8 mM CaCl₂, pH 6.2) and homogenized and aDounce homogenizer in 20 ml of 10 mM Tris-HCl, pH 7.5, 5 mM MgCl₂containing 0.1 mM PMSF, 0.25 μg/ml Leupeptin, 0.5 μg/ml Aprotinin, and 1mM EDTA. Almost no HRS was found in the culture supernatant; all rHRSwas found in cytoplasmic extracts of the infected cells.

Production of HRS in Other Non-mammalian Host Cells

Other methods of producing recombinant HRS in non-mammalian host cellsare also anticipated. For example, cDNA encoding HRS can be ligated intoa yeast expression plasmid, such as pYES2 (Invitrogen, San Diego,Calif.). Following ligation by standard methods known to those of skillin the art, the pYES2 plasmid is transfected into yeast strain INVSc1(MATα his3-Δ1 leu2 trp1-289ura3-52) from Invitrogen (San Diego, Calif).Following incubation, the expressed protein is then isolated by standardmethods from the INVSc1 cells. Other vectors and their correspondingnon-mammalian host cells are also expected to advantageously producerHRS.

Analysis of Expressed Protein

Proof that the infected cell cultures produced authentic HRS wasobtained in the following ways:

(1) immunoprecipitation of both labelled and unlabelled cellsupernatants with monospecific antibodies from sera of polymyositispatients demonstrated a single band which migrated in the identicalposition as HRS immunoprecipitated from normal human tissue culturedcell lines; (2) an enormous rise in activity of HRS in cell linesinfected with recombinant virus but not in cell lines incubated withwild-type baculovirus, nor in uninfected cell lines similarly incubatedand extracted; (3) removal of this new enzymatic activity by adsorptionwith agarose beads bound to monospecific antibodies to HRS but not boundto normal immunoglobulin. Enzymatic activity was measured by the methodof Biswas et al. (Journal of Immunological Methods, 98 (1987) 235-241).

ELISA Assays Using Recombinant HRS

A specific ELISA assay for the presence of anti-HRS antibodies wasdeveloped using rHRS produced by the previously discussed baculovirusexpression system. rHRS was not further purified and therefore, acontrol antigen used for all experiments consisted of similarly preparedprotein preparations from wild type baculovirus infected cells. Optimalconditions for this assay were determined empirically using sera knownto be positive and negative for antibodies against HRS (anti-Jo-1) byimmunoprecipitation.

A crude form of HRS was obtained from baculovirus infected cells and theconcentration of HRS was determined to be 200 μg/ml. Antigen and controlprotein extracts were diluted in buffer and incubated at roomtemperature for at least 2 hours, or at 4° C. overnight, on Immulon 1®flat bottom plates. The plates were then blocked with a blocking agent10 mg/ml Bovine Serum Albumin and incubated at room temperature for atleast 2 hours or at 4° C. overnight. The plates were then washed threetimes in PBS+Tween-20 0.1% and twice in distilled H₂ O.

Serum diluted in buffer with blocking agent was added, and incubated, atroom temperature for 2 hours. The plates were washed again and goatanti-human IgG conjugated to alkaline phosphatase diluted 1:2000 inbuffer with the blocking agent was added. The plates were incubated atroom temperature for another 2 hours, washed, and substrate was added atroom temperature. Optical density measurements were then obtained.

Optimal binding of rHRS with the minimal amount of nonspecificbaculoviral protein background binding was obtained using a buffer ofPBS at pH 7.2 and blocking with Bovine Serum Albumin (BSA) at 10 mg/ml.Using plates prepared in this way, a positive signal was obtained fromsome anti-Jo-1 positive sera diluted 1:64,000. This dilution is fargreater than would be detectable with HRS purified from HeLa cells. Inprevious experiments, serum dilutions of greater than 1:12800 wereunable to detect binding to Hela cell purified HRS (Targoff et al.(1987) J. Immun. 138: 2874-2882). For this reason, the present inventionprovides an improved assay for determining patients with HRS-relatedautoimmune diseases.

To test the binding specificity of anti-Jo-1 to rHRS we compared serafrom myositis positive and negative patients. Nine known anti-Jo-1positive patients were found to have antibodies in their sera that boundthe rHRS antigen. When the same type of ELISA was performed on eightanti-Jo-1 negative myositis patients, three patients with SLE or otherautoimmune diseases, and three normal controls, no binding affinitygreater than that observed in the control wild type baculoviral proteinswas found. This indicated that the rHRS protein was as specific as thepurified HeLa cell HRS and could be used to test for autoimmune diseasesin humans.

In additional experiments, we discovered that the rHRS not only had morebinding affinity for anti-Jo-1 antibodies, but was also more stable thanpurified HRS.

Stability of rHRS

HRS purified from HeLa cells is only stable for a few hours afterisolation (Biswas et al. Federation of European Biochemical Societies(1988) 229(1): 203-205). This instability presents problems indeveloping an efficient assay since fresh HRS isolations were necessaryfor every ELISA. For this reason, it would provide a tremendousadvantage to have a protein with similar antigenic sites as HRS, buthaving an increased stability. Unexpectedly, we discovered that therecombinant HRS is much more stable than HeLa cell purified HRS.

The following ELISAs illustrate the increased stability of rHRS incomparison to purified HRS. Immulon 1® microtiter plates were used inthe experiment. Recombinant HRS was first diluted as described in Tables2 and 3 with PBS. 50λ/well of diluted rHRS was incubated for 2 hours atroom temperature. The wells were aspirated and 100λ/well of PBS-BSA (10mg/ml) was added, then incubated for 4 hours at room temperature. Thewells were washed again with PBS-tween three times, followed by tworinses with H₂ O. 50λ/well of the TEST SERUM 97 (from a polymyositispatient) diluted by the indicated factors in PBS-BSA was added to eachwell and incubated for 2 hours at room temperature. After incubation,the wells were washed three times with PBS-tween and twice with H₂ O. 50λ of goat anti-human-Ig (conjugated to Alkaline Phosphatase) was diluted1:2000 in PBS-BSA and added to each well for 2 hours at roomtemperature. The wells were again washed three times in PBS-tween andtwice in H₂ O. 100λ substrate (4-nitrophenyl phosphate BM 726923) wasdiluted as directed by the manufacturer and added to each well. TheOD₄₀₅ of each well was then read in a microtiter plate reader.

In the first part of the experiment, freshly prepared rHRS was theantigen (Table 2). In the second part of this experiment, rHRS wasstored as a cytoplasmic extract of baculovirus-infected cells for 2weeks at -80° C. (Table 3). In the third part of this experiment, rHRSwas stored after being bound to the microtiter plate at -80° C. for twoweeks (Table 4). As illustrated by the similar results in Tables 2-4,there was very little degradation in the binding affinity of anti-Jo-1serum to the rHRS after storage for two weeks at -80° C.

Furthermore, plates stored an additional two weeks at -20° C. or at +4°C. showed very little reduction in sensitivity for detecting anti-Jo-1antibodies from a positive serum. Amazingly, ELISAs performed on rHRSstored for two months at +4° C. had lost no further sensitivity tobinding with positive sera. We anticipate that rHRS will be similarlystable at room temperature (+25° C.). These results differ dramaticallyfrom similarly performed experiments wherein HRS isolated from HeLacells was stored at -80° C. After storage for less than 72 hours theamount of enzymatic activity in the HeLa cell isolates was substantiallyreduced (Biswas et al. Federation of European Biochemical Societies(1988) 229 (1): 203-205).

For these reasons, the recombinant HRS provides a much better proteinfor assays to detect autoimmune diseases. It can be appreciated thatother methods known to those of skill in the art could also be used withthe rHRS produced in baculovirus to determine binding to anti-Jo-1antibodies. These methods, for instance, could include a radioimmuneassay (RIA), Western Blot or any other method of determiningantibody/antigen binding known to those of skill in the art. From thisinvention it is now possible to make the ELISA plates well in advance ofperforming the experiment. This provides a tremendous commercialadvantage over previous ELISAs that had unstable purified HeLa cell HRS.

                  TABLE 2                                                         ______________________________________                                        TEST SERUM 97                                                                 Not Stored Prior to ELISA                                                     ______________________________________                                        Antibody                                                                             Antigen Dilution                                                       Dilution                                                                             1:500   1:1000    1:2000                                                                              1:4000                                                                              1:8000                                                                              1:16000                            ______________________________________                                        1:1000 0.429   0.415     0.308 0.129 0.113 0.077                              1:2000 0.429   0.270     0.308 0.123 0.069 0.065                              1:4000 0.354   0.351     0.213 0.104 0.075 0.064                              1:8000 0.278   0.236     0.248 0.086 0.069 0.064                              1:16000                                                                              0.196   0.161     0.132 0.090 0.067 0.062                              1:32000                                                                              0.136   0.178     0.155 0.104 0.070 0.060                              1:64000                                                                              0.139   0.104     0.088 0.071 0.062 0.060                              ______________________________________                                        Controls                                                                      Ag         AG + NS   NS      97 TS 686 TS                                                                              PBS                                  ______________________________________                                        0.066      0.076     0.059   0.059 0.061 0.060                                ______________________________________                                         Ag = rHRS, NS = normal serum, 97 TS = serum from polymyositis patient, 68     TS also is serum from polymyositis patient, PBS = Phosphatebuffered           saline.                                                                  

                  TABLE 3                                                         ______________________________________                                        TEST SERUM 97                                                                 Stored for 2 weeks at -80° C.                                          ______________________________________                                        Antibody                                                                             Antigen Dilution                                                       Dilution                                                                             1:500   1:1000    1:2000                                                                              1:4000                                                                              1:8000                                                                              1:16000                            ______________________________________                                        1:1000 0.427   0.335     0.177 0.100 0.083 0.071                              1:2000 0.640   0.298     0.165 0.095 0.063 0.061                              1:4000 0.589   0.249     0.139 0.075 0.064 0.062                              1:8000 0.139   0.126     0.085 0.071 0.070 0.062                              1:16000                                                                              0.283   0.177     0.111 0.072 0.063 0.063                              1:32000                                                                              0.170   0.124     0.094 0.070 0.067 0.066                              1:64000                                                                              0.173   0.104     0.076 0.064 0.062 0.059                              ______________________________________                                        Controls                                                                      Ag         AG + NS   NS      97 TS 686 TS                                                                              PBS                                  ______________________________________                                        0.068      0.075     0.060   0.063 0.063 0.061                                ______________________________________                                         Ag = rHRS = Mixture of Antigen and rHRs, NS = normal serum, 97 TS = serum     from polymyosins patient, 686 TS also is serum from polymyositis patient,     PBS = Phosphatebuffered saline                                           

                  TABLE 4                                                         ______________________________________                                        TEST SERUM 97                                                                 Stored for 2 weeks on microtiter plates at -80° C.                     ______________________________________                                        Antibody                                                                             Antigen Dilution                                                       Dilution                                                                             1:500   1:1000    1:2000                                                                              1:4000                                                                              1:8000                                                                              1:16000                            ______________________________________                                        1:1000 0.451   0.410     0.279 0.151 0.123 0.080                              1:2000 0.421   0.325     0.265 0.119 0.107 0.080                              1:4000 0.386   0.314     0.210 0.131 0.092 0.072                              1:8000 0.311   0.228     0.240 0.118 0.084 0.076                              1:16000                                                                              0.249   0.252     0.205 0.122 0.076 0.079                              1:32000                                                                              0.213   0.147     0.135 0.092 0.071 0.064                              1:64000                                                                              0.130   0.108     0.093 0.089 0.071 0.063                              ______________________________________                                        Controls                                                                      Ag         AG + NS   NS      97 TS 686 TS                                                                              PBS                                  ______________________________________                                        0.069      0.086     0.064   0.067 0.064 0.063                                ______________________________________                                         Ag = rHRS = Mixture of Antigen and rHRs, NS = normal serum, 97 TS = serum     from polymyositis patient, 686 TS also is serum from polymyositis patient     PBS = Phosphatebuffered saline                                           

After performing the above experiments that demonstrated the increasedstability of the rHRS protein, we conducted experiments to determinewhich fragments of the rHRS peptide were antigenically active.

Determination of Antigenic Fragments of rHRS

Fragments of the rHRS protein were produced by protein synthesis in anApplied Biosystems protein synthesizer by well known methods. Fivepeptide fragments were used in competitive inhibitory assays todetermine their ability to block anti-Jo-1 serum binding to rHRS onELISA plates. The following five fragments of rHRS were prepared:

    ______________________________________                                        Fragment Number  Amino Acids                                                  ______________________________________                                        1                1-30                                                         2                14-45                                                        3                31-60                                                        4                1-60                                                         5                1-47                                                         ______________________________________                                    

Samples containing 0.25-500 ng of each protein fragment and anti-Jo-1serum were incubated at room temperature and then added to wellspreviously coated with rHRS. Positive binding between the anti-Jo-1serum and the plate-bound rHRS would indicate that the test fragment wasunable to bind the anti-Jo-1 antibodies. Negative binding between theanti-Jo-1 serum and the plate-bound rHRS would indicate that the testfragment was able to bind the anti-Jo-1 antibodies and competitivelyinhibit the ELISA.

In this experiment, Peptide fragments 4 (amino acids 160) and 5 (aminoacids 1-47) were able to block the interaction of anti-Jo-1 positiveserum with recombinant HRS at fragment concentrations of 250-500ng/well. In control experiments, a complete length rHRS peptide was, asexpected, able to block binding. Amino acid fragments 1, 2 and 3 wereunable to detectably block anti-Jo-1 binding in this assay up to aconcentration of 500 ng/well.

As an extension of the previously discussed method, we bound the fivepeptide fragments onto ELISA plates to directly test their binding toanti-Jo-1 positive serum. Peptides 1-5 were placed in wells at2.5-25,000 ng/well and blocked with 10 mg/ml BSA. Antiserum 97(anti-Jo-1 positive) was added followed by standard-ELISA protocols. Asexpected from the previous data Fragments 1 and 2 were negative.Fragment 3 was weakly positive, while Fragments 4 and 5 were stronglypositive. We have therefore determined that amino acid rHRS Fragments 3,4 or 5 can be used as antigens in an ELISA assay to detect anti-Jo-1positive serum. ELISAs using these antigenic fragments therefore presenta way of detecting the presence of an autoimmune disease in a human. Itcan be appreciated that other methods known to those of skill in the artcould also be used with the inventive peptide fragments to determinebinding to anti-Jo-1 antibodies. These methods, for instance, couldinclude a radioimmune assay (RIA), Western Blot or any other method ofdetermining antibody/antigen binding known to those of skill in the art.

It is also anticipated that kits can be developed to detect binding ofautoimmune antibodies with rHRS produced in non-mammalian host cells.These kits contain, for instance, recombinant Histidyl tRNA synthetase,a vessel for performing the binding assay, and reagents for detectingbinding of rHRS to the autoimmune antibodies. Vessels could bemicrotiter plates, tubes, bottles, or any other container for performinga binding assay. The reagents for detecting binding could be, forexample, ELISA reagents such as labeled goat anti-human antibodies,along with the proper buffers for performing an ELISA assay. ELISAs arewell known methods by those with skill in the art. A large number ofother sets of reagents are known for use in the large number of suitableimmunoassays known to those having ordinary skill in the art. Theappropriate regents for each of these immunoassays can be readilyascertained by those having ordinary skill in the art For example,"Immunochemical Assays and Biosensor Technology for the 1990's")Nakamura et al (eds), American Society for Microbiology (1992), thedisclosure of which is hereby incorporated by reference, lists a largenumber of isotopic and nonisotopic immunoassays that can be adapted fordetection of HRS antigen within the scope of the present invention. Thereagents necessary for any particular immunoassay can be readilydetermined by those having ordinary skill in the art. Generally, thesereagents will include a label, such as a radionuclide, enzyme,fluorescent label, luminescent label, vesicle label or particle label.Appropriate additional reagents for detection of the label are requiredfor detection of some of these labels, such as a substrate for an enzymelabel or an agglutination product, such as erythrocytes, latex orgelatin, for particle labels.

It can be appreciated that the scope of the present invention should notbe limited by the previous examples, but is defined by the followingclaims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 24                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                         (iv) ANTI-SENSE: NO                                         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nucleic acid                                                       (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      AGTCTCTGTTCCACGATGGAGAA23                                                     (2) INFORMATION FOR SEQ ID NO:13:                                             ( i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 24                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CCACCATGGTTGCTGGACATAGTC 24                                                   (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      ACACTGCCCACACCCAGGGGCTC 23                                                    (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      ACAC TGCCCACANCCANGGGCTC23                                                    (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      TGTCCAAGCTTGTCTACTGAG21                                                       (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                       (iii) HYPOTHETICAL: NO                                                       (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      TATCCAGCTTGTCTACTGAG20                                                        (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      CTTGGGGGTTTTGAGCACAAATT23                                                     (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22                                                                (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      CCACTTGAGCCGCCTGCTGTCT22                                                      (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 ( A) LENGTH: 20                                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GGCTACTAAGGGAACTTGGG20                                                        (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 491 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: internal                                                   (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: Eco                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                       AlaLysAsnIleGlnAlaIleArgGlyMetAsnAspTyrLeuProGly                             151015                                                                        GluThrAlaIleTrpGlnArgIleGluGlyThrLeuLysAsnVal Leu                             202530                                                                        GlySerTyrGlyTyrSerGluIleArgLeuProIleValGluGlnThr                              354045                                                                         ProLeuPheLysArgAlaIleGlyGluValThrAspValValGluLys                             505560                                                                        GluMetTyrThrPheGluAspArgAsnGlyAspSerLeuThrLeuArg                               65707580                                                                     ProGluGlyThrAlaGlyCysValArgAlaGlyIleGluHisGlyLeu                              8590 95                                                                       LeuTyrAsnGlnGluGlnArgLeuTrpTyrIleGlyProMetPheArg                              100105110                                                                     HisGluArgProGlnXaaXaaLysGlyArgTyrArgGlnPh eHisGln                             115120125                                                                     LeuGlyCysGluValPheGlyLeuGlnGlyProAspIleXaaAspAla                              130135140                                                                      GluLeuIleMetLeuThrAlaArgTrpTrpArgAlaLeuGlyIleSer                             145150155160                                                                  GluXaaHisValThrLeuGluLeuAsnSerIleXaaGlyS erLeuGlu                             165170175                                                                     AlaArgAlaAsnTyrArgAspAlaLeuValAlaPheLeuGluGlnHis                              180185 190                                                                    LysGluLysLeuAspXaaXaaXaaXaaGluAspCysLysArgArgMet                              195200205                                                                     TyrThrAsnXaaXaaXaaXaaXaaXaaXaaProLeu ArgValXaaXaa                             210215220                                                                     XaaXaaXaaLeuAspSerLysAsnProGluValGlnAlaLeuLeuAsn                              225230235 240                                                                 AspAlaProAlaLeuGlyAspTyrLeuAspGluGluSerArgGluHis                              245250255                                                                     PheAlaGlyLeuCysLysLeuLeuGluSer AlaGlyIleAlaTyrThr                             260265270                                                                     ValAsnGlnArgXaaXaaXaaXaaXaaXaaLeuValArgGlyLeuAsp                              275280 285                                                                    TyrTyrAsnArgThrValPheGluTrpValThrAsnSerLeuGlySer                              290295300                                                                     GlnXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXa aXaaXaaXaaXaa                             305310315320                                                                  XaaXaaXaaXaaXaaXaaXaaGlyThrValCysAlaGlyGlyArgTyr                              325 330335                                                                    AspGlyLeuValGluGlnLeuGlyGlyArgAlaThrXaaXaaXaaXaa                              340345350                                                                     XaaXaaProAlaValGlyPheA laMetGlyLeuGluArgLeuValLeu                             355360365                                                                     LeuValGlnXaaXaaXaaXaaAlaValAsnProGluPheLysAlaAsp                              370375 380                                                                    ProValValAspIleTyrLeuValAlaSerGlyAlaAspThrSerGln                              385390395400                                                                  AlaAlaMetAlaLeuAlaGlu ArgLeuArgXaaXaaAspGluLeuXaa                             405410415                                                                     XaaProGlyValLysLeuXaaMetThrAsnHisGlyGlyGlyAsnPhe                              420 425430                                                                    LysLysGlnPheAlaArgAlaAspLysTrpGlyAlaArgValAlaVal                              435440445                                                                     ValLeuGlyGluSerGlu ValAlaAsnGlyThrAlaValValLysAsp                             450455460                                                                     LeuXaaXaaXaaXaaArgSerGlyGluGlnThrAlaValAlaGlnAsp                              465470 475480                                                                 SerValAlaAlaHisLeuArgThrLeuLeuGly                                             485490                                                                        (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 561 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: Yea                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      MetSerSerAlaThrAlaXaaXaaXaaXaaXaaX aaXaaXaaXaaXaa                             151015                                                                        XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaAlaAlaThrSerAlaPro                              2025 30                                                                       ThrAlaAsnAlaAlaAsnXaaXaaAlaLeuLysAlaSerLysAlaPro                              354045                                                                        LysLysGlyLysLeuGlnValSerLeuLysThr ProLysGlyThrLys                             505560                                                                        AspTrpAlaAspSerAspMetValIleArgGluAlaIlePheSerThr                              657075 80                                                                     LeuSerGlyLeuPheLysLysHisGlyGlyValThrIleAspThrPro                              859095                                                                        ValPheGluLeuArgGluIleLeuAlaG lyLysTyrGlyGluAspSer                             100105110                                                                     XaaXaaXaaXaaLysLeuIleTyrAsnLeuGluAspGlnGlyGlyGlu                              115120 125                                                                    LeuCysSerLeuArgTyrAspLeuThrValProPheAlaArgTyrVal                              130135140                                                                     AlaMetAsnAsnIleXaaXaaXaaGlnSerIle LysArgTyrHisIle                             145150155160                                                                  AlaLysValTyrArgArgAspGlnProAlaMetThrLysGlyArgMet                              165 170175                                                                    ArgGluPheTyrGlnCysAspPheAspValAlaGlyThrPheGluSer                              180185190                                                                     MetValProAspSerGluCys LeuSerIleLeuValGluGlyLeuThr                             195200205                                                                     SerLeuGlyIleLysAspPheLysIleLysLeuAsnHisArgLysIle                              2102 15220                                                                    LeuAspGlyIlePheGlnIleAlaGlyValLysAspGluAspValArg                              225230235240                                                                  LysIleSerSerAlaValAs pLysLeuAspLysSerProTrpGluAla                             245250255                                                                     ValLysLysGluMetThrGluGluLysGlyGlnSerGluGluThrAla                              260 265270                                                                    AspLysIleGlyGluTyrValLysLeuAsnGlySerLeuLysGluIle                              275280285                                                                     HisAlaValLeuSerA laAspAlaAsnIleThrSerXaaXaaAsnGlu                             290295300                                                                     LysAlaLysGlnGlyLeuAspAspIleXaaAlaThrLeuMetLysTyr                              305310 315320                                                                 ThrGluAlaPheAspIleAspSerPheIleSerPheAspLeuSerLeu                              325330335                                                                     AlaArgGly LeuAspTyrTyrThrGlyLeuIleTyrGluValValThr                             340345350                                                                     SerAlaSerAlaProProGluAsnAlaSerGluLeuLysLysLysAla                               355360365                                                                    LysSerAlaGluAspAlaSerGluPheValGlyValGlySerIleAla                              370375380                                                                     AlaGlyGlyArgTyr AspAsnLeuValAsnMetPheSerGluAlaSer                             385390395400                                                                  GlyLysLysSerThrGlnIleProCysValGlyIleSerPheGlyVal                               405410415                                                                    GluArgIlePheSerLeuIleLysGlnArgIleAsnSerXaaSerThr                              420425430                                                                     Th rIleLysProThrAlaThrXaaGlnValPheValMetAlaPheGly                             435440445                                                                     GlyGlyLysAspTrpThrGlyTyrXaaLeuProGluArgMetLysVal                               450455460                                                                    ThrLysGlnLeuTrpAspAlaGlyIleGluAlaGluTyrValTyrLys                              465470475480                                                                  A laLysAlaAsnProArgLysGlnPheAspThrThrLysLysAlaGly                             485490495                                                                     CysHisIleAlaValIleLeuGlyLysGluGluTyrLeuGluGlyLy s                             500505510                                                                     LeuArgValLysArgLeuGlyGlnGluPheAlaAspAspAspGlyGlu                              515520525                                                                      LeuValSerAlaAlaAspIleValProIleValGlnGluLysLeuSer                             530535540                                                                     GlnIleHisGluAspGlyLeuAsnGluValThrArgLeuIleLysGly                               545550555560                                                                 Leu                                                                           (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 560 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                    (iii) HYPOTHETICAL: NO                                                       (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: Hum                                                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      MetAlaGluArgAlaAlaLeuGluGluLeuValLysLeuGlnGlyGlu                              15 1015                                                                       ArgXaaValArgGlyLeuLysGlnGlnLysAlaSerAlaGluLeuIle                              202530                                                                        GluGluGluValAlaLysLeuLeuL ysLeuLysAlaGlnLeuGlyPro                             354045                                                                        AspGluSerLysGlnLysPheValLeuLysThrProLysGlyThrArg                              5055 60                                                                       AspTyrSerProArgGlnMetAlaValArgGluLysValPheAspVal                              65707580                                                                      IleIleArgCysPheLysArgHisGly AlaGluValIleAspThrPro                             859095                                                                        ValPheGluLeuLysGluThrLeuMetGlyLysTyrGlyGluAspSer                              100 105110                                                                    XaaXaaXaaXaaLysLeuIleTyrAspLeuLysAspGlnGlyGlyGlu                              115120125                                                                     LeuLeuSerLeuArgTyrAspLeu ThrValProPheAlaArgTyrLeu                             130135140                                                                     AlaMetAsnLysLeuXaaXaaXaaThrAsnIleLysArgTyrHisIle                              145150 155160                                                                 AlaLysValTyrArgArgAspAsnProAlaMetThrArgGlyArgTyr                              165170175                                                                     ArgGluPheTyrGlnCy sAspPheAspIleAlaGlyAsnPheAspPro                             180185190                                                                     MetIleProAspAlaGluCysLeuLysIleMetGluIleLeuSerSer                              195 200205                                                                    LeuGlnIleGlyAspPheLeuValLysValAsnAspArgArgIleLeu                              210215220                                                                     AspGlyMetPheAlaIleCysG lyValSerAspSerLysPheArgThr                             225230235240                                                                  IleCysSerSerValAspLysLeuAspLysValSerTrpGluGluVal                               245250255                                                                    LysAsnGluMetValGlyGluLysGlyLeuAlaProGluValAlaAsp                              260265270                                                                     ArgIleGly AspTyrValGlnGlnHisGlyGlyValSerLeuValGlu                             275280285                                                                     GlnLeuLeuXaaGlnAspProLysLeuSerGlnXaaXaaAsnLysGln                              290 295300                                                                    AlaLeuGluGlyLeuGlyAspLeuXaaLysLeuLeuPheGluTyrLeu                              305310315320                                                                  ThrLeuPhe GlyIleAspAspLysIleSerPheAspLeuSerLeuAla                             325330335                                                                     ArgGlyLeuAspTyrTyrThrGlyValIleTyrGluAlaValLeuXaa                               340345350                                                                    XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaLeuGlnThrProAlaGln                              355360365                                                                     AlaGl yGluGluProLeuXaaXaaXaaGlyValGlySerValAlaAla                             370375380                                                                     GlyGlyArgTyrAspGlyLeuValGlyMetPheXaaAspProLysGly                              385 390395400                                                                 ArgLysXaaXaaXaaValProCysValGlyLeuSerIleGlyValGlu                              405410415                                                                      ArgIlePheSerIleValGluGlnArgLeuGluAlaLeuGluGluLys                             420425430                                                                     IleArgThrThrGluThrXaaGlnValLeuValAlaSerXaaXaaAl a                             435440445                                                                     GlnLysLysLeuLeuGluXaaXaaXaaXaaGluArgLeuLysLeuVal                              450455460                                                                     Ser GluLeuTrpAspAlaGlyIleLysAlaGluLeuLeuTyrLysLys                             465470475480                                                                  AsnProLysLeuLeuAsnGlnLeuGlnTyrCysGluGluAlaGlyI le                             485490495                                                                     ProLeuValAlaIleIleGlyGluGlnGluLeuLysAspGlyValIle                              500505 510                                                                    LysLeuArgSerXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa                              515520525                                                                     ValThrSerArgGluGluValAspValArgArgGluAspLeu XaaXaa                             530535540                                                                     ValGluGluIleLysArgArgThrGlyGlnProLeuCysIleCysXaa                              545550555 560                                                                 (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 561 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE: N-terminal                                                 (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: Ham                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                     MetAlaSerXaaProAlaLeuGluGluLeuValLeuAsnSerArgHis                              151015                                                                        ArgLeuValArgGlyLeuLysGlnGlnLys AlaSerAlaAspGlnIle                             202530                                                                        GluGluGluValAlaLysLeuLeuLysLeuLysAlaGlnLeuGlyHis                              3540 45                                                                       AspGluSerLysGlnLysPheValLeuLysThrProLysGlyThrArg                              505560                                                                        AspTyrSerProArgGlnMetAlaValArgGluLysV alPheAspVal                             65707580                                                                      IleIleCysCysPheLysArgHisGlyAlaGluValIleAspThrPro                              85 9095                                                                       ValPheGluLeuLysGluThrLeuMetGlyLysTyrGlyGlnAspCys                              100105110                                                                     XaaXaaXaaXaaLysLeuIleTyrAs pLeuLysAspGlnGlyGlyGlu                             115120125                                                                     LeuLeuSerLeuArgTyrAspLeuThrValProPheGlyArgTyrLeu                              130135 140                                                                    AlaMetAsnAsnLeuXaaXaaXaaThrAsnIleLysArgTyrHisIle                              145150155160                                                                  AlaLysValTyrArgArgAspAsnP roAlaMetThrArgGlyArgTyr                             165170175                                                                     LeuAsnSerIleThrValAspPheAspIleAlaGlyGlnPheAspPro                              180 185190                                                                    MetIleProAspAlaGluCysLeuLysIleMetCysGluIleLeuSer                              195200205                                                                     SerLeuGlnIleGlyLysPhe LeuValLysValAsnAspArgArgIle                             210215220                                                                     LeuAspGlyMetPheAlaValCysGlyValProAspSerLysPheArg                              225230 235240                                                                 ThrIleCysSerSerValAspLysLeuAspLysValSerTrpGluGlu                              245250255                                                                     ValLysAsnGluMet ValGlyGluLysGlyLeuAlaProGluValAla                             260265270                                                                     AspArgIleGlyAspTyrValGlnGlnHisGlyGluValCysLeuVal                              275 280285                                                                    GluGlnLeuLeuXaaGlnAspProLysLeuSerGlnXaaXaaAsnLys                              290295300                                                                     GlnAlaValGluGlyLeuGl yAspLeuXaaLysLeuLeuPheGluTyr                             305310315320                                                                  LeuThrLeuPheGlyIleAspAspLysIleSerPheAspLeuSerLeu                               325330335                                                                    AlaArgGlyLeuAspTyrTyrThrGlyValIleTyrValAlaValLeu                              340345350                                                                     XaaXaaX aaXaaXaaXaaXaaXaaXaaXaaXaaLeuGlnMetProThr                             355360365                                                                     GlyAlaGlyGluGluProTrpXaaXaaXaaXaaCysGlyGlnCysGly                              370 375380                                                                    CysTrpArgArgTyrAspGlyLeuValGlyMetPheXaaAspProLys                              385390395400                                                                  GlyArg LysXaaXaaXaaValProCysValGlyLeuSerIleGlyVal                             405410415                                                                     GluArgIlePheSerIleValGluGlnArgLeuGluAlaLeuGluGlu                               420425430                                                                    LysValArgThrThrGluThrXaaGlnValLeuValAlaSerXaaXaa                              435440445                                                                     Ala GlnLysLysLeuAlaGlyXaaXaaXaaXaaGlyGluThrLysAla                             450455460                                                                     CysLeuGlnLeuTrpAspAlaGlyIleLysAlaGluLeuLeuTyrLys                              465 470475480                                                                 LysAsnProLysLeuLeuAsnGlnLeuGlnTyrCysGluGluThrGly                              485490495                                                                      IleProLeuValAlaIleIleGlyGluGlnGluLeuLysAspGlyVal                             500505510                                                                     IleLysLeuArgSerXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa Xaa                             515520525                                                                     XaaValAlaSerArgGluGluValAspValArgArgGluAspLeuXaa                              530535540                                                                     X aaValGluGluIleArgArgArgThrAsnGlnProLeuTyrValCys                             545550555560                                                                  Xaa                                                                       

We claim:
 1. A method for determining the presence of an autoimmunedisease in a mammal comprising:isolating a sample of body fluid from amammal, said body fluid containing antibodies; contacting said isolatedbody fluid with recombinant Histidyl tRNA synthetase produced byexpression of a mammalian Histidyl tRNA synthetase transfected into hostinsect cells, said recombinant Histidyl tRNA synthetase beingsubstantially free of endogenous mammalian Histidyl tRNA synthetaseimmunoreactivity produced by said cells; and detecting the presence orabsence of binding of said antibodies to said recombinant Histidyl tRNAsynthetase, wherein detectable binding of said antibodies to saidrecombinant Histidyl tRNA synthetase indicates the presence of anautoimmune disease in said mammal.
 2. The method of claim 1, whereinsaid body fluid is blood serum.
 3. The method of claim 1, wherein saidautoimmune disease is selected from the group consisting of myositis,polymyositis and dermatomyositis.
 4. The-method of claim 1, whereinprior to said contacting step, said recombinant Histidyl tRNA synthetaseis stored for greater than 24 hours.
 5. The method of claim 1, whereinsaid contacting and detecting steps comprises an enzyme-linkedimmunosorbent assay (ELISA).
 6. The method of claim 4, wherein saidHistidyl tRNA is stored at a temperature within the range -80° C. to 25°C.
 7. A kit for determining the presence of antibodies from anautoimmune disease in the blood serum of a mammal comprising:recombinanthistidyl tRNA synthetase produced by expression of an oligonucleotidecoding for mammalian histidyl tRNA synthetase transfected into hostinsect cells, wherein said recombinant histidyl tRNA synthetase issubstantially free from endogenous mammalian histidyl tRNA synthetaseimmunoreactivity produced by said host cells; a vessel for performing anassay to detect binding of said antibodies to said recombinant HistidyltRNA synthetase; reagents for detecting the binding of said antibodiesto said recombinant Histidyl tRNA synthetase, wherein positive bindingindicates the presence of an autoimmune disease in said mammal.
 8. Thekit of claim 7, wherein said vessel is a microtiter plate.
 9. The kit ofclaim 7, wherein said reagents for detecting the binding of saidantibodies to said recombinant Histidyl tRNA synthetase comprise labeledanti-human antibodies.
 10. The kit of claim 7, wherein said reagents fordetecting comprise reagents for performing an ELISA.